LBHBOs are those hydrogen bonds in which the distance between donor and acceptor atoms has decreased, causing the barrier against proton transfer to fall to approximately the zero-point energy of the hydrogen atom. LBHBOs are stronger than ordinary H-bonds; however, the hydrogen atom is not found to be equidistant between donor and acceptor atoms, unlike single-well H-bonds. The mechanism by which serine proteases catalyze the hydrolysis of amides has received much attention from mechanistic and structural enzymologists, and the role of the aspartic acid in the catalytic traid has been not entirely clear. Recently, it has been demonstrated that LBHB forms between the conserved histidine and aspartic acid residues of the catalytic traid of chymotrysin and proposed that the low barrier hydrogen bond facilitates analysis. The presence of a LBHB in serine proteases is supported by model chemistry. Yet the presence of LBHBOs a the active-sites of enzymes remains controversial. We found a biological peak at 18.1-18.4 ppm in the 1H NMR of Subtilisin Carlsberg (SC) at pH 5.5, consistent with previous work and consistent with the presence of a LBHB. We determined the fractionation factor of this resonance using 1H NMR to be 0.6 at pH 5.5 and 0.75 at pH 6. This value indicates a preference for hydrogen over deuterium and between SC and the slow-binding inhibitors Z-leucylalanylphenylalanyltriflouromethuleketone and z-LLF-CF3 are 18.8-19.0 ppm, implying the existence of a stronger hydrogen bond between His and Asp than at low pH. This peak is narrower and more Lorentzian than in free SC. The fractionation factor of this peak is 0.86, a result which underscores the fact that fractionation factors are sensitive to other influences besides LBHBOs.